Chemokines constitute a family of small cytokines that are produced in inflammation and regulate leukocyte recruitment (Baggiolini, M. et al., xe2x80x9cInterleukin-8 and related chemotactic cytokinesxe2x80x94CXC and CC chemokines,xe2x80x9d Adv. Immunol. 55: 97-179 (1994); Springer, T. A., xe2x80x9cTraffic signals on endothelium for lymphocyte recirculation and leukocyte emigration,xe2x80x9d Annu. Rev. Physiol. 57: 827-872 (1995); and Schall, T. J. and K. B. Bacon, xe2x80x9cChemokines, leukocyte trafficking, and inflammation,xe2x80x9d Curr. Opin. Immunol. 6: 865-873 (1994)). Chemokines are capable of selectively inducing chemotaxis of the formed elements of the blood (other than red blood cells), including leukocytes such as neutrophils, monocytes, macrophages, eosinophils, basophils, mast cells, and lymphocytes, such as T cells and B cells. In addition to stimulating chemotaxis, other changes can be selectively induced by chemokines in responsive cells, including changes in cell shape, transient rises in the concentration of intracellular free calcium ions ([Ca2+]i), granule exocytosis, integrin upregulation, formation of bioactive lipids (e.g., leukotrienes) and respiratory burst, associated with leukocyte activation. Thus, the chemokines are early triggers of the inflammatory response, causing inflammatory mediator release, chemotaxis and extravasation to sites of infection or inflammation.
Two subfamilies of chemokines, designated as CXC and CC chemokines, are distinguished by the arrangement of the first two of four conserved cysteine residues, which are either separated by one amino acid (as in CXC chemokines IL-8, xcex3IP-10, Mig, PF4, ENA-78, GCP-2, GROxcex1, GROxcex2, GROxcex3, NAP-2, NAP-4) or are adjacent residues (as in CC chemokines MIP-1xcex1, MIP-1xcex2, RANTES, MCP-1, MCP-2, MCP-3, I-309). Most CXC chemokines attract neutrophil leukocytes. For example, the CXC chemokines interleukin 8 (IL-8), platelet factor 4 (PF4), and neutrophil-activating peptide 2 (NAP-2) are potent chemoattractants and activators of neutrophils. The CXC chemokines designated Mig (monokine induced by gamma interferon) and IP-10 (xcex3IP-10, interferon-gamma inducible 10 kDa protein) are particularly active in inducing chemotaxis of activated peripheral blood leukocytes. CC chemokines are generally less selective and can attract a variety of leukocyte cell types, including monocytes, eosinophils, basophils, T lymphocytes and natural killer cells. CC chemokines such as human monocyte chemotactic proteins 1-3 (MCP-1, MCP-2 and MCP-3), RANTES (Regulated on Activation, Normal T Expressed and Secreted), and the macrophage inflammatory proteins 1xcex1 and 1xcex2 (MIP-1xcex1 and MIP-1xcex2) have been characterized as chemoattractants and activators of monocytes or lymphocytes, but do not appear to be chemoattractants for neutrophils.
Chemokines act through receptors which belong to a superfamily of seven transmembrane spanning G-protein coupled receptors (Murphy, P. M., xe2x80x9cThe molecular biology of leukocyte chemoattractant receptors,xe2x80x9d Annu. Rev. Immunol., 12: 593-633 (1994); Gerard, C. and N. P. Gerard, xe2x80x9cThe pro-inflammatory seven transmembrane segment receptors of the leukocyte,xe2x80x9d Curr. Opin. Immunol., 6: 140-145 (1994)). This family of G-protein coupled (serpentine) receptors comprises a large group of integral membrane proteins, containing seven transmembrane-spanning regions. The receptors are coupled to G proteins, which are heterotrimeric regulatory proteins capable of binding GTP and mediating signal transduction from coupled receptors, for example, by the production of intracellular mediators. Two of these receptors, the interleukin-8 (IL-8) receptors, IL-8R1 (interleukin-8 receptor type 1; Holmes, W. E. et al., xe2x80x9cStructure and functional expression of a human interleukin-8 receptor,xe2x80x9d Science, 253: 1278-1280 (1991)) and IL-8R2 (interleukin-8 receptor type 1; Murphy, P. M. and H. L. Tiffany, xe2x80x9cCloning of complementary DNA encoding a functional human interleukin-8 receptor,xe2x80x9d Science, 253: 1280-1283 (1991)), are largely restricted to neutrophils and recognize the NH2-terminal Glu-Leu-Arg (ELR) motif, an essential binding epitope in those CXC chemokines that induce neutrophil chemotaxis (Clark-Lewis, I. et al., xe2x80x9cStructure-activity relationships of interleukin-8 determined using chemically synthesized analogs. Critical role of NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis, exocytosis, and receptor binding activities,xe2x80x9d J. Biol. Chem., 266: 23128-23134 (1991); Hxc3xa9bert, C. A. et al., xe2x80x9cScanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding,xe2x80x9d J. Biol. Chem., 266: 18989-18994 (1991); and Clark-Lewis, I. et al., xe2x80x9cPlatelet factor 4 binds to interleukin 8 receptors and activates neutrophils when its N terminus is modified with Glu-Leu-Arg,xe2x80x9d Proc. Natl. Acad. Sci. USA, 90: 3574-3577 (1993)). Five distinct CC chemokine receptors have been described, and are designated CC-CKR1, -2, -3, -4 and -5 (CC-CKR, CC chemokine receptor; Neote, K. et al., xe2x80x9cMolecular cloning, functional expression, and signaling characteristics of a CC chemokine receptor,xe2x80x9d Cell, 72: 415-425 (1993); Gao, J.-L. et al., xe2x80x9cStructure and functional expression of the human macrophage inflammatory protein 1xcex1/RANTES receptor,xe2x80x9d J. Exp. Med., 177: 1421-1427 (1993); Charo, I. F. et al., xe2x80x9cMolecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails,xe2x80x9d Proc. Natl. Acad. Sci. USA, 91: 2752-2756 (1994); Myers, S. J., et al., J. Biol. Chem., 270: 5786-5792 (1995); Combadiere, C. et al., Cloning and functional expression of a human eosinophil CC chemokine receptor,xe2x80x9d J. Biol. Chem., 270 (27): 16491-16494 (1995); and Correction, J. Biol. Chem., 270: 30235 (1995); Ponath, P. D. et al., xe2x80x9cMolecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils,xe2x80x9d J. Exp. Med., 183: 2437-2448 (1996); and Daugherty, B. L. et al., xe2x80x9cCloning, expression, and characterization of the human eosinophil eotaxin receptor,xe2x80x9d J. Exp. Med., 183: 2349-2354 (1996); Power, C. A. et al., 1995, xe2x80x9cMolecular cloning and functional expression of a novel CC chemokine receptor cDNA from a human basophilic cell line,xe2x80x9d J. Biol. Chem., 270: 19495-19500 (1995); Hoogewerf, A. J. et al.,xe2x80x9d Molecular cloning of murine CC CKR-4 and high affinity binding of chemokines to murine and human CC CKR-4,xe2x80x9d Biochem. Biophys. Res. Commun., 218: 337-343 (1996); Samson, M. et al., xe2x80x9cMolecular cloning and functional expression of a new human CC-chemokine receptor gene,xe2x80x9d Biochemistry, 35: 3362-3367 (1996)). The CC chemokine receptors occur on several types of leukocytes, including monocytes, granulocytes and lymphocytes, and recognize CC chemokines, but not CXC chemokines.
In contrast to monocytes and granulocytes, lymphocyte responses to chemokines are not well understood. Notably, none of the receptors of known specificity appear to be restricted to lymphocytes and the chemokines that recognize these receptors cannot, therefore, account for events such as the selective recruitment of T lymphocytes that is observed in T cell-mediated inflammatory conditions. Moreover, although a number of proteins with significant sequence similarity and similar tissue and leukocyte subpopulation distribution to known chemokine receptors have been identified and cloned, the ligands for these receptors remain undefined. Thus, these proteins are referred to as orphan receptors. The characterization of the ligand(s) of a receptor, is essential to an understanding of the interaction of chemokines with their target cells, the events stimulated by this interaction, including chemotaxis and cellular activation of leukocytes, and the development of therapies based upon modulation of receptor function.
The present invention relates to proteins or polypeptides, referred to herein as isolated and/or recombinant mammalian (e.g., a primate such as a human) IP-10/Mig receptor proteins designated CXC Chemokine Receptor 3 (CXCR3) and variants thereof. Recombinant CXCR3 proteins and variants can be produced in host cells as described herein. In one embodiment, a CXCR3 protein or variant thereof is characterized by selective binding (e.g., high affinity binding) of one or more chemokines, such as IP-10 and/or Mig, and/or the ability to induce a (one or more) cellular response(s) (e.g., chemotaxis, exocytosis, release of one or more inflammatory mediators).
Another aspect of the present invention relates to isolated and/or recombinant nucleic acids which encode a mammalian (e.g., a primate such as a human) CXCR3 protein or variant thereof. The invention further relates to recombinant nucleic acid constructs, such as plasmids or retroviral vectors, which contain a nucleic acid which encodes a protein of the present invention or a variant thereof. The nucleic acids and constructs can be used to produce recombinant receptor proteins and host cells comprising a construct. In another embodiment, the nucleic acid encodes an antisense nucleic acid which can hybridize with a second nucleic acid encoding a CXCR3 protein and which, when introduced into cells, can inhibit the expression of receptor.
Antibodies reactive with CXCR3 receptors can be produced using the proteins or variants thereof (e.g., a peptide) or cells expressing receptor protein or variant as immunogen, for example. Such antibodies or fragments thereof are useful in therapeutic, diagnostic and research applications, including the purification and study of the receptor proteins, identification of cells expressing surface receptor, and sorting or counting of cells.
Also encompassed by the present invention are methods of identifying ligands of the receptor, inhibitors (e.g., antagonists) or promoters (e.g., agonists) of receptor function. In one embodiment, suitable host cells which have been engineered to express a receptor protein or variant encoded by a nucleic acid introduced into said cells are used in an assay to identify and assess the efficacy of ligands, inhibitors or promoters of receptor function. Such cells are also useful in assessing the function of the expressed receptor protein or polypeptide.
According to the present invention, ligands, inhibitors and promoters of receptor function can be identified in a suitable assay, and further assessed for therapeutic effect. Inhibitors of receptor function can be used to inhibit (reduce or prevent) receptor activity, and ligands and/or promoters can be used to induce (trigger or enhance) normal receptor function where indicated. Thus, the present invention provides a method of treating inflammatory diseases including autoimmune disease and graft rejection, comprising administering an inhibitor of receptor function to an individual (e.g., a mammal). The present invention further provides a method of stimulating receptor function by administering a ligand or promoter to an individual, providing a new approach to selective stimulation of leukocyte function, which is useful, for example, in the treatment of infectious diseases and cancer.